Tag Archives: Ultrafree-DA

Performed nested RACE PCR on the RACE PCR products generated on 20110722 using the following nested primers: PGS2_ngspRACE_5′ (SR ID: 1350) and PGS2_ngspRACE_3′ (SR ID: 1349). Removed 2uL from each of the primary PCR reactions and brought up to 100uL in tricine EDTA (supplied in the Clontech SMARTer RACE cDNA Amplification Kit). Performed the nested RACE PCR according to the Clontech manual. Briefly, this is the same as the primary RACE PCR reaction, but using 5uL of the diluted primary PCR product and 1uL of the Nested Universal Primer (instead of 5uL of the 10X Universal Primer Mix). Master mix calcs and set up are here. Cycling params followed “Program 2″ of the Clontech protocol, modified for nested primers, and are as follows:

20 cycles:

94C 30s

68C 30s

72C 3m

Results:

Gel Layout:

Lane 1 – Hyperladder 1

Lanes 2-6 = 5′ RACE Library

Lane 2 – nGSP1 (5′ RACE primer)

Lane 3 – nGSP2 (3′ RACE primer)

Lane 4 – Neg. Control (no RACE primers)

Lane 5 – Neg. Control (nGSP1, no Universal primer)

Lane 6 – Neg. Control (nGSP2, no Universal primer)

Lane 7 – Empty

Lanes 8-12 = 3′ RACE Library

Lane 8 – nGSP1 (5′ RACE primer)

Lane 9 – nGSP2 (3′ RACE primer)

Lane 10 – Neg. Control (no RACE primers)

Lane 11 – Neg. Control (nGSP1, no Universal primer)

Lane 12 – Neg. Control (nGSP2, no Universal primer)

First of all, we see the appropriate response of each primer only producing amplicons in their respective libraries (i.e. 5′ primer only works in 5′ RACE library). This simply confirms that the primers were designed correctly. Secondly, our negative controls are clean. Thirdly, we get distinct bands from both primers. The bands marked with blue arrows in the image above were excised and purified using Ultrafree DA spin columns (Millipore). These products will be used for cloning and eventual sequencing.

Due to the failure of the primary PCR on both 5′ and 3′ RACE cDNA libraries (from 20080619) yesterday, will perform nested PCR using a nested GSP designed by Steven (CgPGSRACEsrNGSP1; SR ID:1209). The 5uL of PCR reactions that were set aside yesterday were diluted to 250uL with tricine-EDTA (supplied with the Clontech SMARTer RACE cDNA Amplification Kit) as instructed in the Clontech manual. The master mix and tube layouts were exactly the same as yesterday’s, but instead of using 2.5uL of RACE cDNA library as template, I used 5uL of the diluted PCR reaction. Additionally, Universal Nested Primers were used instead of the Universal Primer Mix (both supplied in the kit). Cycling parameters followed “Program 2″ from the Clontech manual for 25 cycles.

Entire PCR rxns were run on a 1.2% gel, as instructed by the Clontech manual.

Results:

So….. What we see here is a melted gel!

But! We also see a successful PCR! The first three lanes (excluding the Hyperladder I) are 5′ RACE rxn, followed by two different negative controls (the negative control in the last lane is the one we’re really concerned with and it’s totally clean). The next three lanes are the 3′ RACE rxn, followed by two different negative controls (the negative control in the last lane is the one we’re really concerned with and it’s totally clean). As hoped/expected, we got a nice, clear product in the 5′ RACE rxn.

The bright band (~1500bp) in the 5′ RACE rxn PCR was excised and purified using Millipore Ultrafree DA columns according to protocol. Will clone and sequence this product.

MM07 – Fails to produce any bands of any size. Suggests the presence of intron(s) causing the size of the potential amplicon to exceed the capabilities of the polymerase under these cycling conditions.

MM08 – Produces a band of ~400bp which is well below the expected 1540bp (if no introns) size. Due to the faintness of the band, the band was not excised. Will consult with Steven to see if he thinks it worth repeating to produce sufficient product for sequencing.

MM09 – Produce a ~500bp band. The band was excised. This band size is ~275bp larger than the expected size of 225bp. This implies the presence of an intron in this region. This band size differs from that produced by MM10, which suggests that this primer set can be used for qPCR AND distinguish between the COX1 and COX2 isoforms.

MM10 – Produced a ~700bp band. The band was excised. This band size is ~475bp larger than the expected size of 225bp. This implies the presence of an intron in this region. This band size differs from that produced by MM09, which suggests that this primer set can be used for qPCR AND distinguish between the COX1 and COX2 isoforms.

MM11 – Produced multiple bands, of which two were excised; a ~3000bp band and a ~600bp band. These bands were excised solely based on their intensity and their immediate useability for sequencing. Will discuss with Steven on whether or not this should be repeated and the other bands excised for sequencing purposes. Both bands that were excised exceed the expected band size of ~275bp, suggesting the presence of multiple introns. Additionally, the presence of so many products suggests that the primers are not very specific, in regards to their target.

MM12 – An extremely faint band of ~350bp can be seen, however, due to it’s faintness and it’s small size (expected size was ~812bp), the band was not excised. Will discuss with Steven to see if this warrants repeating to accumulate sufficient product for sequencing purposes. No amplification of any larger products suggests the presence of introns, causing the size of the potential amplicon to exceed the capabilities of the polymerase under these cycling conditions. This is also confirmed by the MM11 PCR results in which a 3000bp band was produced. Since the primer set in MM12 has an additional 600bp at the 5′ end, this has already exceeded the abilities of the polymerase, even if this addtional 600bp does NOT include an additional intron. However, it is curious that the MM12 primer set does not produce smaller, spurious PCR products as is seen in the MM11 primer set (these two primer sets both use the same forward primer).

This was repeated to generate more PCR product for sequencing purposes. PCR master mix calcs and cycling params are here. Master mixes 04 and 05 (MM04 and MM05) were repeated to gain more PCR product from the faint 550bp & 1500bp bands(MM04) and 5000bp band (MM05).

Samples on the left portion of the gel are from the MM04 primer combo and those on the right are from the MM05. Boxed bands were excised, purified using Millipore Ultra DA-free spin columns and stored @ -20C in Sam’s “Misc. -20C Box.”

Interestingly in the MM05 set, inconsistent, faint bands of ~400-500bp are visible. These were not visible the first time this PCR was conducted (see 20110118), but the exposure of the gel image wasn’t turned up as high as in this image. Due to their inconsistency and extremely low yield, these bands were not excised.

Bioline Hyperladder I used for marker. Gel is loaded with template samples at the far left of each master mix group with two no template controls (NTC) in the remaining two wells of each master mix group. All NTCs on the gel are clean.

All bands surrounded by a green box were excised from the gel.

MM01, MM02 and MM03 – The smallest expected band (i.e. no intron present) would have been 1000bp in MM01. Instead, we see faint banding of multiple sizes less than 1000bp in both MM01 and MM02. MM03 fails to produce any bands. This potentially suggests a couple of things. Firstly, the multiple banding produced in MM01 and MM02 suggests that the PCR conditions lead to some non-specific priming and should be optimized. Secondly, the fact that no bands were produced that are equal to or larger than the “no intron size” suggests that intron(s) may exist in the 5′ region of the COX gene and are large enough that the polymerase had insufficient time/ability to amplify.

MM04 – Three distinct bands were produced: 2000bp, 1500bp and 550bp. The size of band that would have been produced had an intron NOT been present would have been ~550bp. A band of this size was produced in this PCR reaction. However, two additional bands were produced. The presence of these two larger bands lends additional evidence for the existence of multiple isoforms of COX (which is also supported by the fact that multiple isoforms of COX are known to exist in most other species). The 2000bp band was excised and purified with Millipore Ultra-free DA spin columns and stored @ -20C until a sequencing plate is readied.

MM05 – A distinct band of ~5000bp was produced. This is significantly larger than the “no intron size” of ~1130bp, suggesting the presence of an intron. This band was excised, but not purified due to the low concentration of DNA in the gel. The gel slice was stored @ -20C until this PCR reaction could be repeated to accumulate sufficient product for sequencing.

MM06 – A distinct band of ~2200bp was produced. This is significantly larger than the “no intron size” of ~620bp, suggesting the presence of an intron. The band was excised and purified with Millipore Ultra-free DA spin columns and stored @ -20C until a sequencing plate is readied.

The PCR reactions reveal the presence of intron(s) in the COX gene we’re investigating as well as providing evidence for the existence of multiple isoforms in C.gigas. Since the PCR products that have been excised for sequencing are so large, additional primers will need to be designed closer to the introns in order to generate smaller products that can be fully sequenced. Additionally, all reactions using the primer designed to anneal in the 5’UTR of COX (SR ID: 1150) failed to produce useful results. This is either due to poor performance of the primer under these reaction conditions or due to the presence of a large intron in the 5′ region of the gene. Additional reverse primers will be designed that anneal closer to the 5′ portion of the COX gene in hopes of characterizing the 5′ genomic sequence better.

After speaking with Steven today about the potential existence/”discovery” of multiple isoforms, he decided to map the newly-released C.gigas 454 NGS data to the existing COX coding sequence in GenBank (FJ375303). The alignment is shown below.

The two 454 reads that map closest to the 5′ end of the COX coding sequence match up nearly perfectly, with periodic SNPs. The remaining 454 reads that map to the COX coding sequence are very different and provide very good evidence of a previously unidentified isoform of COX in C.gigas. Primers will be designed from both the existing COX sequence in GenBank (FJ375303) and the other potential isoform. These primers will likely be used in both qPCR and for sequencing purposes, in order to be able to distinguish and characterize both isoforms. Additionally, BLASTing will be performed with the sequences from both isoforms to evaluate how they match up with existing COX isoforms in other species.

Samples were submitted for sequencing. Mac prepped all Roberts Lab samples excluding the Opsin VMC gel slice 2 from 20091217-02. The gel slice was purified with Millipore spin columns. The sample was diluted 1:1 with H2O and submitted for sequencing, one time from each direction using the Sep_Op_Fw2/Rv2 primers. Plate layout can be found here on sheet labeled “20091223”.

This is a repeat of yesterday’s set up with LABY primers, but with an annealing temp of 53C in hopes of improving the number of amplicons generated from additional samples. See yesterday’s PCR run for info on samples.

Results: Samples were loaded 1-29 and three negative controls from left to right, top to bottom.

The lower annealing temperature clearly resulted in more products. The ~500bp band was cut from each lane and stored @ -20C. All bands will be purified using Millipore spin columns and then sent for sequencing.

This was done on the numbered tubes using the LABY A/Y primers for eventual sequencing. Turns out many of the tubes have some info (other than just a number) on their sides which might provide more information regarding which isolate they actually are. PCR set up is here. Annealing temp 55C.

Tube-#

Side Label

1

VA1423-1

2

VA1423 2CB

3

VA1423-3

4

VA-1423 4

5

VA1423-6 6

6

VA1423-10

7

VA1423-12

8

VA1423-15

9

VA1423-26

10

VA1423-28

11

VA1423-290 2003 Isolate

12

VA1423 29 2004 Isolate

13

VA-1423-33

14

VA1423-37

15

XMAC13T

16

X-MAC-19T

17

XMAC 20T

18

X-MAD 10T

19

X-MAD-14T

20

X-MAD-18T

21

XMAE 11T

22

XMAE 13T

23

BC05CA8T

24

BC05CA 15T

25

BC05CA 18T

26

BC05CA 20T

27

98 MFS 61A

28

CRT W 1HE/H11

29

CRSH 5B3

Results: Samples were loaded 1-29 and three negative controls from left to right, top to bottom.

There are four prominent bands from Tubes 23, 27, 28, 29. These four bands were excised and purified with Millipore spin columns according to protocol. They will be sent for sequencing. There are faint bands visible from Tubes 9 & 11. Due to the faintness, they were not excised as there may not be enough product for sequencing. The remainder of the samples failed to produce any amplicons.

This is a repeat of yesterday’s PCR due to the presence of bands in the water-only samples. Will use reagents and universal 16s bacterial primers (27F & 1492R) provide by the Horner-Devine lab in hopes of: 1) getting this two work and, 2) figuring out the source of the contamination.

All rxns were prepared sterily and all instruments, racks, tubes, tips and water were UV-sterilized for ~45mins in the biological hood. Rxns were prepared in the biological hood. PCR setups are here. Anneal 60C. Cycling params same as yesterday.

Lane 1 – 100bp ladder

Lane 2 – DNA (HD Rxn 1)

Lane 2 – H2O (HD Rxn 1)

Lane 3 – H2O (HD Rxn 1)

Lane 4 – DNA (HD Rxn 2)

Lane 5 – H2O (HD Rxn 2)

Lane 6 – H2O (HD Rxn 2)

Lane 7 – DNA (SR Rxn)

Lane 8 – H2O (SR Rxn)

Lane 9 – H2O (SR Rxn)

Lane 10 – 100bp ladder

Results: Well, we got our band and NO contamination in any H2O lanes. The super-bright, 1500bp band will be excised and purified using Millipore spin columns and submitted for sequencing. However, this gel is interesting because the primers provided by Mike (used in HD Rxn 1 and SR Rxn) did not amplify anything…